Omalizumab (XolairTM) is a recombinant monoclonal anti-IgE antibody used in the fight against severe allergic asthma that generates $500 million per year. However, 2 out of every 1,000 patients treated with omalizumab suffer from anaphylaxis, a severe allergic reaction to the therapeutic. Moreover, omalizumab immunotherapy is prohibitively expensive, reaching nearly $30,000 per year. Thus, there is a great unmet need for an improved omalizumab composition that is affordable to health care consumers and tolerated in circulation. Genentech currently produces omalizumab in mammalian cell culture, which is expensive and susceptible to viral contamination. Alternatively, Escherichia coli culture is inexpensive, well-characterized, fast-growing, and not susceptible to viral contamination. However, E. coli is not typically used for full-length antibody production and instead is relegated to expression of smaller, engineered antibodies such as antigen binding fragments (Fabs). Fabs bind equally or better to target antigens, but their persistence in the human body can be limited. In spite of these shortcomings, therapeutic antibody fragments are regularly produced in E. coli including Genentech's ranibizumab (LucentisTM), a Fab designed for intraocular use. An emerging solution to prolong the half-life of Fabs in circulation is the covalent attachment of human-type oligosaccharides. Lipid-linked oligosaccharides identical to human blood group ABO O-type antigens are naturally synthesized in E. coli strains of serogroup O86. The hypothesis of this proposal is that non-pathogenic strains of E. coli can be engineered to produce and transfer O-type antigens to specific sites in recombinant Fabs. To test this hypothesis, the objective of this proposal is to generate anti-IgE recombinant antibodies with improved serum tolerance in by: (i) cloning and expressing the biosynthetic machinery for the human blood group O-type oligosaccharide in E. coli K12, (ii) cloning and expressing a recombinant anti-IgE Fab in E. coli, and (iii) conjugating blood group type O-type antigens to anti-IgE Fabs in glycoengineered E. coli. It is anticipated that these studies will result in an efficient E. coli expression platform for the production of anti-IgE Fabs linked to human blood group O-type oligosaccharides in a controlled, rapid, and cost-effective manner. These studies are significant because they explore a paradigm-shifting technology for the production of therapeutic Fabs for the treatment of asthma and other immunological diseases. PUBLIC HEALTH RELEVANCE: Monoclonal antibodies are prominent therapeutics in the fight against many immunological diseases including severe allergic asthma. Unfortunately, the production process for monoclonal antibodies is expensive, resulting in a cost of therapy that is unaffordable for the healthcare consumer. The proposed studies focus on producing well-tolerated therapeutic antibody conjugates in Escherichia coli fermentation without the need for costly mammalian cell culture or in vitro chemical modification.